The mammalian pyruvate dehydrogenase complex (PDC) connects glycolysis to oxidative metabolism and fatty acid synthesis with the irretrievable loss of glucose carbons. PDC is regulated by the pyruvate dehydrogenase (E1) component being inter-converted between an active (non-phosphorylated) form and an inactive (phosphorylated) form. To achieve tissue-specific control of glucose utilization, PDC is regulated by at least four pyruvate dehydrogenase kinase isoforms. Kinase activities are greatly enhanced and modified in their primary effector control upon binding to the dihydrolipoyl acetyltransferase (E2), a four-domain protein that organizes as a 60mer to form the central framework of the complex. Lipoyl domains, two in E2's outer structure and one in a dihydrolipoyl dehydrogenase-binding protein (E3BP), bind the kinases and serve as molecular switches in their regulatory control. Using an all-human system of recombinantly produced components with a focus on kinase isoforms 2 and 3, studies are proposed to dissect how E2 via its lipoyl domains produces the predominant changes in kinase activity. With an expressly designed armament of E2 structure, the PI will evaluate, via analytical ultracentrifuge and activity-based approaches, how the dynamic lipoyl domain interactions with kinase isoforms lead to direct (conformational) activation, facilitate efficient access to the E1 substrate, mediate feedback control (stimulate kinase inactivation of PDC) and modify the responses to effectors that directly bind the kinase to produce feed-forward and energy state control. Important variables manipulated are the lipoyl domain source, its oligomeric state, and the reaction state of its lipoyl group. Mutated and prosthetic group modified forms of the inner lipoyl domain of E2 will be used to determine how this domain is structurally empowered to transform the kinase isoform activities. The long term objective is to understand the tissue-specific control of PDC and to delineate the unique properties of kinase isoforms overexpressed in diabetes which inactivates PDC thereby minimize glucose use.